CHEMICAL HYPOXIA INCREASES CYTOSOLIC CA2-FREE RADICAL FORMATION( AND OXYGEN)

'Chemical hypoxia' was produced in isolated rat hepatocytes. The cells were immobilized in agarose gel threads and perfused with Krebs-Hense leit bicarbonate buffer equilibrated with 95% O-2 + 5% CO2 or 95% air + 5% CO2, During 'chemical hypoxia', 2 mM KCN + 0.5 mM iodoacetate (CN -IAA) were added to the perfusate for 30 min, Cytosolic ionized Ca2+ ( Ca-i(2+)) was measured with aequorin, the formation of oxygen free rad icals by lucigenin-enhanced chemiluminescence and cell injury by the r ate of LDH released by the cells in the effluent perfusate. As soon as the cells were exposed to CN-IAA in the presence of 95% O-2 + 5% CO2, Ca-i(2+) increased rapidly to reach 1.5 mu M within 10 min, and oxyge n free radical formation increased 5-fold. The increase in LDH release was temporally delayed and occurred only during the recovery phase. T he results were not significantly different when the cells were perfus ed with KHB equilibrated with 95% air + 5% CO2, except that oxygen fre e radical formation increased 13-fold. These results suggest that both a rise in Ca-i(2+) and a formation of reactive oxygen species could b e responsible for the cell injury and the cell death induced by CN-IAA poisoning.